Propionic acid, an important three-carbon organic acid, holds significant application value in the food, chemical, and pharmaceutical synthesis industries. Microbial fermentation for propionic acid production has emerged as a crucial research direction in the field of biosynthesis. The wild-type Propionibacterium jensenii strain PA2 was utilized as the progenitor strain to obtain a high-yield P. jensenii strain for propionic acid production. Initial fermentation parameters were established, followed by superimposed space mutagenesis and ethyl methanesulfonate chemical mutagenesis, coupled with high-efficiency screening. Following the superimposed mutagenesis of the PA2 strain, screening was performed to obtain the high-yield DJ04-16 strain, which achieved a propionic acid production of 31.78 g•L−1, representing a 31.27% increase over PA2 output. Through orthogonal optimization, the optimal fermentation medium was determined as: glucose (30 g•L−1), glycerol (35 g•L−1), peptone (9 g•L−1), yeast extract (10 g•L−1), ferrous sulfate (80 mg•L−1), calcium pantothenate (0.8 mg•L−1), biotin (0.8 mg•L−1), magnesium sulfate (160 mg•L−1), dipotassium phosphate (2 g•L−1), monopotassium phosphate (1 g•L−1), and corn steep liquor (8 g•L−1). When cultivated in this optimized medium, DJ04-16 produced 33.75 g•L−1 of propionic acid after 144 h of fermentation. In conclusion, the synergistic combination of space and chemical mutagenesis significantly improves propionic acid productivity in P. jensenii. The developed mutant DJ04-16 exhibits considerable potential for industrial-scale application, thereby providing a robust technological platform for microbial propionic acid biosynthesis.